Identification and characterization of human bone-derived cells

et al. 2018

Int J of Rheum Dis

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Aim: Ankylosing spondylitis (AS) is characterized by excessive spinal ankylosis and bone formation. Alkaline phosphatase (ALP) activity is reported to be high in AS, but little is known about the molecular relationship between ALP and AS. The aims of this study were to investigate the relevance of ALP to AS and the role of ALP in the regulation of osteoblast differentiation in AS. Methods: High-throughput data with accession numbers GSE73754 and GSE41038were downloaded from the Gene Expression Omnibus. We retrospectively collected and compared the ALP levels of male patients with AS to those of sex-and agematched healthy controls (HC) and rheumatoid arthritis (RA) patients. Total serum ALP and ALP activity were measured in the AS and RA groups. ALP gene expression and intracellular ALP activity were analyzed in microarray data from primary diseases control (Ct) and AS-bone-derived cells (BdCs) and in vitro experiments. Furthermore, the effect of ALP inhibitor was examined in both primary Ct-and AS-BdCs under osteoblast differentiation. Regulation of runt-related transcription factor 2 (RUNX2) by ALP was also analyzed. Results: Alkaline phosphatase level was higher in AS compared with RA and HC and was associated with radiograph progression. ALP expression was also enriched in the bone tissue of AS patients. Furthermore, AS-BdCs exhibited increasing ALP activity, leading to accelerated osteoblastic activity and differentiation. Intriguingly, inhibition of ALP reduced RUNX2 expression, a master transcriptional factor in osteoblasts, and differentiation status of both primary Ct-and AS-BdCs. Treatment of ALP activator or inhibitor modulated RUNX2 protein level and RUNX2 regulated ALP promoter activity, indicating a reciprocal ALP-RUNX2 positive feedback to regulate osteoblast differentiation. Conclusion: Alkaline phosphatase was highly expressed in AS patients, may be involved in the ankylosis of AS, and represents a possible therapeutic target for ankylosis. K E Y W O R D S alkaline phosphatase, ankylosing spondylitis, ankylosis, osteoblastic activity, osteoblastic differentiation

Section: Methodsmentioning

confidence: 99%

“…Experiments were performed using a microarray system (Macrogen Inc, Seoul, Korea) that has previously been described in detail …”

Section: Methodsmentioning

confidence: 99%

Regulation of osteoblasts by alkaline phosphatase in ankylosing spondylitis

Han

et al. 2018

Int J of Rheum Dis

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“…Once cells reached about 90% confluency, growth media were replaced with differentiation media containing osteogenic inductive agents. Osteoprogenitors were incubated with osteoinductive agents (50 µM ascorbic acid (AA), 10 mM β-glycerolphosphate, and 100 nM dexamethasone) for osteoblast differentiation, as described [36,37]. Differentiation media were changed every 3 days.…”

Section: Isolation Of Primary Osteoprogenitors Osteoblasts Osteoblamentioning

confidence: 99%

“…Microarray data were previously reported [36], and differentially expressed genes were screened by Wnt/β-catenin signaling and its related molecules were reanalyzed and presented in this study. All microarray analyses and visualization were conducted using R.3.4.1.…”

Section: Microarray Datamentioning

confidence: 99%

DKK1 Induced by 1,25D3 Is Required for the Mineralization of Osteoblasts

Yoon

et al. 2020

Cells

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1α,25-dihydroxyvitamin D3 (1,25D3), the most popular drug for osteoporosis treatment, drives osteoblast differentiation and bone mineralization. Wnt/β-catenin signaling is involved in commitment and differentiation of osteoblasts, but the role of the Dickkopf-related protein 1 (DKK1), a Wnt antagonist, in osteoblasts remains unknown. Here, we demonstrate the molecular mechanism of DKK1 induction by 1,25D3 and its physiological role during osteoblast differentiation. 1,25D3 markedly promoted the expression of both CCAAT/enhancer binding protein beta (C/EBPβ) and DKK1 at day 7 during osteoblast differentiation. Interestingly, mRNA and protein levels of C/EBPβ and DKK1 in osteoblasts were elevated by 1,25D3. We also found that C/EBPβ, in response to 1,25D3, directly binds to the human DKK1 promoter. Knockdown of C/EBPβ downregulated the expression of DKK1 in osteoblasts, which was partially reversed by 1,25D3. In contrast, overexpression of C/EBPβ upregulated DKK1 expression in osteoblasts, which was enhanced by 1,25D3. Furthermore, 1,25D3 treatment in osteoblasts stimulated secretion of DKK1 protein within the endoplasmic reticulum to extracellular. Intriguingly, blocking DKK1 attenuated calcified nodule formation in mineralized osteoblasts, but not ALP activity or collagen synthesis. Taken together, these observations suggest that 1,25D3 promotes the mineralization of osteoblasts through activation of DKK1 followed by an increase of C/EBPβ.

“…Osteogenic, chondrogenic, and ligamentous differentiation. Osteogenic differentiation methods and activity assessments have been previously reported 36 . Briefly, cells from the distal and middle ACL regions were seeded in growth medium (DMEM with high glucose, Hyclone, SH30243.01) and then differentiated with osteogenic medium (ascorbic acid, dexamethasone, and β-glycerophosphate) into mature osteoblasts.…”

Section: Methodsmentioning

confidence: 99%

Anterior cruciate ligament remnant cells have different potentials for cell differentiation based on their location

et al. 2020

Sci Rep

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Histological and cytological observations of the human anterior cruciate ligament (ACL) had been described, but the differentiation potency based on their location is still unknown. To determine and compare proliferation and differentiation potential of cells derived from distal and middle thirds of the ACL remnant, ACL remnant was initially marked at the distal third (within 10 mm from the tibial insertion) and middle third (between 10-20 mm from the tibial insertion) and then dissected. Both the middle and distal third regions of ACL remnant were analyzed using CD34 + cell counting. Cell proliferation rate did not differ in both middle and distal third regions of ACL remnant, but they showed different characteristics in cell differentiation depending on their location. The distal third region of the ACL remnant had a tendency for chondrogenic differentiation with higher expression of CD34 + cells. On the other hand, the middle third region of ACL remnant had a strong tendency for osteogenic and ligamentous differentiation. Characteristics of the ACL remnant tissues should be considered when performing remnant-preserving or harvesting ACL remnants for tissue engineering. Anterior cruciate ligament reconstruction (ACLR) is one of the most common surgical procedures in the field of orthopaedic sports medicine, with more than 130,000 procedures performed annually in the United States alone 1,2. It has been well documented that a completely ruptured ACL does not spontaneously heal because of poor vascular supply and an unfavourable intra-articular environment 3. Given the importance of its biomechanical function, surgical treatment is generally accepted as the standard procedure for restoring knee stability. In most cases, non-augmented primary repair has been unsuccessful, and therefore ACL reconstruction is required 1,4,5. For surgical success, ACLR requires tendon graft healing in a surgically created bone tunnel and maturation (i.e., ligamentization) of the graft substance 4,6-9. Indeed, the lack of vascularity within the tendon graft induces degeneration or micro ruptures during the early postoperative period 10. To overcome these issues, tissue engineering using stem cells has been widely explored as a means to achieve early graft healing, tendon regeneration, and bone integration. Recently, reports have shown that ruptured human ACL tissues can possess numerous vascular-derived stem cells and that ACL-derived CD34 + cells can promote healing and have high expansion and multilineage differentiation potential 7,11-13. Mifune et al. 14 demonstrated that ACL-derived CD34 + cells contributed to tendon-bone healing after ACLR via angiogenesis and osteogenesis enhancements 15. Furthermore, Matsumoto et al. 16 found that incorporation of ruptured ACL tissues in autologous grafts reduced tunnel enlargement in ACLR 16,17. However, with regard to their clinical application, potential advantages of remnant-derived stem cells are still questionable. Histological observations of the uninjured human ACL have shown a differe...